The invention relates to the field of medicine and biotechnology. More particularly, the invention relates to the use of a recombinant model antigen DNA vaccine incorporating tandem copies (as, for example linear concatamers) of the synthetic peptides derived from the CR2 binding motif of the C3d domain of the third component (C3) of the human complement system. Furthermore, this inventions relates to a method and kit for the implementation of said method of such a construct as a vaccine against malaria infections and as a treatment of the same.
Background on the Malarial Disease
Malaria currently represents one of the most prevalent infections in tropical and subtropical areas throughout the world. Per year, malaria infections lead to severe illnesses in hundreds of million individuals worldwide, while it kills 1 to 3 million people, primarily in developing and emerging countries every year. The widespread occurrence and elevated incidence of malaria are a consequence of the increasing numbers of drug-resistant parasites and insecticide-resistant parasite vectors. Other factors include environmental and climatic changes, civil disturbances, and increased mobility of populations.
Malaria is caused by the mosquito-borne hematoprotozoan parasites belonging to the genus Plasmodium. Four species of Plasmodium protozoa (P. falciparum, P. vivax, P. ovale and P. malariae) are responsible for the disease in humans; many others cause disease in animals, such as P. yoelii and P. berghei in mice. P. falciparum accounts for the majority of infections and is the most lethal type (“tropical malaria”). Malaria parasites have a life cycle consisting of several stages. Each stage is able to induce specific immune responses directed against the corresponding occurring stage-specific antigens.
Malaria parasites are transmitted to man by several species of female Anopheles mosquitoes. Infected mosquitoes inject the “sporozoite” form of the malaria parasite into the mammalian bloodstream. Sporozoites remain for a few minutes in the circulation before invading hepatocytes. At this stage, the parasite is located in the extra-cellular environment and is exposed to antibody attack, mainly directed to the “circumsporozoite” (CS) protein (CSP), a major component of the sporozoite surface. Once in the liver, the parasites replicate and develop into so-called “schizonts.” These schizonts occur in a ratio of up to 20,000 per infected cell. During this intra-cellular stage of the parasite, main players of the host immune response are T-lymphocytes, especially CD8+ T-lymphocytes (Bruna-Romero O., 2001A). After about one week of liver infection, thousands of so-called “merozoites” are released into the bloodstream and enter red blood cells, becoming targets of antibody-mediated immune response and T-cell secreted cytokines. After invading erythrocytes, the merozoites undergo several stages of replication and transform into so-called “trophozoites” and into schizonts and merozoites, which can infect new red blood cells. This stage is associated with overt clinical disease. A limited amount of trophozoites may evolve into “gametocytes,” which is the parasite's sexual stage. When susceptible mosquitoes ingest erythrocytes, gametocytes are released from the erythrocytes, resulting in several male gametocytes and one female gametocyte. The fertilization of these gametes leads to zygote formation and subsequent transformation into ookinetes, then into oocysts, and finally into salivary gland sporozoites.
Malaria Vaccines Incorporating CSP Antigens
Current strategies for developing effective malaria vaccines are to elicit 1) humoral responses against sporozoite surface antigen (Ag) to reduce infection of the liver, 2) cellular responses against hepatic stages to kill infected liver cells and reduce the subsequent blood stage infection, 3) humoral responses against erythrocytic stage Ag to eliminate residual infection and disease, and 4) humoral responses against sexual stage Ag to reduce transmission. (Wolfgang W. Leitner *. M., 1997). One candidate for inclusion is the circumsporozoite protein (CSP) which is present in three developmental stages, including sporozoites and infected hepatocytes (Nussenzweig. V., 1989) and exoerythrocytic merozoites, but not erythrocytic merozoites (Atkinson, 1989).
The CS protein is the only P. falciparum antigen demonstrated to consistently prevent malaria when used as the basis of active immunization in humans against mosquito-borne infection, albeit at levels that are often insufficient. Theoretical analysis has indicated that the vaccine coverage, as well as the vaccine efficiency, should be above 85% or, otherwise, mutants that are more virulent may escape (Clyde D. F., 1973).
The sporozoite stage of malaria parasites carries CSP on its outer surface (Aikawa, 1981) which expresses a unique immunodominant epitope recognized by immunized or repeatedly infected hosts (Zavala F. A., 1983). Sera from mice immunized with Plasmodium berghei sporozoites immunoprecipitate a single 44,000Mr protein, the circumsporozoite (CS) protein (CSP), from extracts of surface-labeled sporozoites (Zavala F. A., 1983). Immunoprecipitation of extracts of metabolically labeled sporozoites with a monoclonal antibody (3D11) directed to the CSP demonstrated that the 44,000Mr membrane form is derived from a 54,000-Mr intracellular precursor (Zavala F. A., 1983). The CSP from monkey and human malaria parasites contains amino and carboxy-terminal regions of relatively low immunogenicity which flank a central region of highly immunogenic, tandemly repeated amino acid units, the sequences of which differ from species to species (Damem, 1984). Monoclonal antibodies to the repeated amino acid units neutralize parasite infectivity (Nussenzweig, 1969), suggesting that CSP might be useful as sporozoite-stage vaccines.
The central region of the CSP contains tandem repetitive peptide sequences that appear to be the dominant targets for Ab responses during infection (Nussenzweig. V., 1989). The N and C-terminal regions flanking the central CSP repeats contain several immunologically and structurally important features, such as MHC class I and II epitopes (Romero, 1990), and peptide structures that appear to be important for sporozoite invasion of hepatocytes (Aley, 1986). CSP-specific immune responses have been induced with various synthetic peptides and fragments or full-length recombinant proteins (Egan, 1987). The responses were B cell (Egan, 1987) or T cell dependent and in the case of T cell responses, were either CD4 or CD8 dependent (Migliorini, 1993). However, only a few of the immunogens induced responses that reduced the infection rate upon challenge (Egan, 1987). While some CSP repeat region-specific mAbs reduce infection rates (Potocnjak, 1980), repeat region-specific polyclonal immune sera have little protective efficacy (Egan, 1987). Immune responses to a sulfatide binding motif from P. falciparum CSP can reduce P. berghei infection rates in vaccinated mice (Chatterjee. S., 1995). CSP-specific CD8+ T cell clones prepared by vaccination with a synthetic peptide reduced infection rates in recipient mice after adoptive transfer, but active immunization with the same peptide did not affect infection rates (Nussenzweig. V., 1989). Vaccination of BALB/c mice with recombinant vaccinia expressing P. berghei CSP induced CTL and reduced the infection rate upon challenge. Thus, as a sporozoite surface Ag, a hepatic stage Ag, and an exoerythrocytic merozoite surface Ag, CSP is a good target for neutralizing Ab as well as cellular immune responses (Wolfgang W. Leitner *. M., 1997).
The C terminus of the circumsporozoite protein (CSP) is anchored to the parasite cell membrane by a glycosylphosphatidylinositol (GPI) glycolipid. This GPI signal sequence functions poorly in heterologous eukaryotic cells, causing CSP retention within internal cell organelles during genetic immunization. Cellular location of antigen has quantitative and qualitative effects on immune responses induced by genetic immunization. Removal of the GPI signal sequence has a profound effect on induction and efficacy of CSP-specific immune response after genetic immunization of BALB/c mice with a gene gun (Sandra Scheiblhofer, 2001). The CSP produced from a plasmid lacking the GPI anchor signal sequence (CSP−A) is secreted and soluble, but that produced by a CSP+A plasmid is not. The CSP−A plasmid induces a highly polarized Th2 type response, in which the CSP-specific IgG antibody titer is three- to fourfold higher, and the protective effect is significantly greater than that induced by a CSP+A plasmid. Thus, engineering plasmid constructs for proper cellular localization of gene products is a primary consideration for the preparation of optimally efficacious DNA vaccines. (Sandra Scheiblhofer, 2001).
During genetic immunization with plasmid DNA, the amount of plasmid-associated CpG motif administered has the strongest effect on the quantitative and qualitative aspects of immune responses (Klinman, 1997). Another important factor is the physical form of the expressed protein (Barry, 1997). Immunization of mice with plasmids expressing secreted soluble antigen induces strong humoral responses of the Th2 type, as measured by induction of IgG1 isotype antibodies, whereas immunization with plasmid expressing cytoplasmic and membrane-anchored antigens tends to induce a more balanced response as measured by induction of IgG2a isotype antibodies (Forms, 1999).
DNA Vaccinations—a Background
DNA vaccination has become the fastest growing field in vaccine technology following reports at the beginning of the 90's that plasmid DNA induces an immune response to the plasmid-encoded antigen (Wolff J A, 1990). However, DNA vaccination in many cases is hampered by poor efficacy. Thus, as discussed below, various strategies must be developed to improve immune responses induced by genetic vaccines.
In contrast to vaccines that employ recombinant bacteria or viruses, genetic vaccines consist only of DNA (as plasmids) or RNA (as mRNA), which is taken up by cells and translated into protein. In case of gene-gun delivery, plasmid DNA is precipitated on to an inert particle (generally gold beads) and forced into the cells with a helium blast. Transfected cells then express the antigen encoded on the plasmid resulting in an immune response. Like live or attenuated viruses, DNA vaccines effectively engage both MHC-I and MHC-II pathways allowing for the induction of CD8+ and CD4+ T cells whereas antigen present in soluble form, such as recombinant protein, generally induces only antibody responses (Wolfgang W. Leitner H. Y., 1999).
Because genetic vaccines are relatively inexpensive and easy to manufacture and use, their immunogenicity and efficacy have been analyzed in a large number of systems and results from preclinical studies have supported human clinical trials. Studies have rapidly moved from small laboratory animals to primates and clinical trials are currently being conducted for diseases such as cancer, HIV-infection, or malaria (Hoffman S L, 1997).
The quick acceptance of genetic vaccines in experimental settings is due to the many advantages this strategy has over traditional vaccines. However, the efficacy of genetic vaccines in many systems has not proven to be satisfactory, leading some to conclude that genetic vaccines are not a viable alternative to conventional vaccines and will never replace them (Manickan E, 1997). Some studies, however, purport that DNA vaccines are more efficacious than some established vaccines based on recombinant proteins, recombinant viruses, or both (Schirmbeck R, 1996). Indeed, DNA vaccines can circumvent many of the problems associated with recombinant protein-based vaccines, such as high costs of production, difficulties in purification, incorrect folding of antigen and poor induction of CD8+ T cells. DNA also has clear advantages over recombinant viruses, which are plagued with the problems of pre-existing immunity, risk of insertion-muta-genesis, loss of attenuation or spread of inadvertent infection (Restifo N, 1999).
DNA vaccines usually consist of plasmid vectors (derived from bacteria) that contain heterologous genes (transgenes) inserted under the control of a eukaryotic promoter, allowing protein expression in mammalian cells (HL, 1997). An important consideration when optimising the efficacy of DNA vaccines is the appropriate choice of plasmid vector. The basic requirements for the backbone of a plasmid DNA vector are a eukaryotic promoter, a cloning site, a polyadenylation sequence, a selectable marker and a bacterial origin of replication (Gurunathan S, 2000). A strong promoter may be required for optimal expression in mammalian cells. For this, some promoters derived from viruses such as cytomegalovirus (CMV) or simian virus 40 (SV40) have been used. A cloning site downstream of the promoter should be provided for insertion of heterologous genes, and inclusion of a polyadenylation (polyA) sequence such as the bovine growth hormone (BGH) or SV40 polyadenylation sequence provides stabilisation of mRNA transcripts. The most commonly used selectable markers are bacterial antibiotic resistance genes, such as the ampicillin resistance gene. However, since the ampicillin resistance gene is precluded for use in humans, a kanamycin resistance gene is often used. Finally, the Escherichia coli ColE1 origin of replication, which is found in plasmids such as those in the pUC series, is most often used in DNA vaccines because it provides high plasmid copy numbers in bacteria enabling high yields of plasmid DNA on purification (Helen S Garmory, 2003).
Genetic vaccines can be delivered into the host by several routes and methods. Needle-injection into muscle tissue and into the skin is the most commonly used method (Wolff J A, 1990). Also the spleen and a variety of mucosal surfaces (Wang B, 1997) including those of the nose and gut have been targeted (Wolfgang W. Leitner H. Y., 1999). Scale-up from small rodents to larger animals and humans may not be an obstacle: A given DNA-dose may effectively induce an immune response regardless of body size (Cox G J, 1993). Despite the large number of genetic vaccine-studies conducted so far, many of the results are difficult to compare and inconsistent. A number of factors determine the magnitude and type of immune response induced by plasmid DNA (see Table in FIG. 1).
Genetic vaccines may mimic some aspects of the natural infection of host-cells. However, microorganisms contain surface-molecules such as LPS and a variety of soluble factors that function as adjuvants, alerting the immune system to ‘danger’ by inducing inflammation. The potency of genetic vaccines may be significantly enhanced by mimicking these signals with synthetic adjuvants such as QS21 or monophosphoryl lipid A (MPL) (Sasaki S, 1997). However, DNA plasmids without adjuvant are able to induce remarkably strong immune responses to the encoded antigen.
Because myocytes are able to take up some of the plasmid injected into the muscle the mechanism of intramuscular immunization seemed very straightforward (see FIG. 2) (Wolfgang W. Leitner H. Y., 1999). However, muscle is not considered an immunologically relevant tissue as myocytes lack the characteristics of antigen-presenting cells (APC) such as MHC-II expression, costimulatory molecules or marked cytokine-secretion. Even the co-expression of costimulatory molecules or cytokines like GM-CSF or IL-12 is insufficient to turn non-hematopoetic cells into efficient APC (Iwasaki A, 1997). Thus, it seems unlikely that muscle tissue is the immune activating component.
Two possible scenarios could explain the mechanism of immune-priming by intramuscularly injected genetic vaccines (see FIG. 2). First, myocytes are the antigen-factories that supply professional APCs with antigen for the induction of an immune response in the form of full-length protein or peptides (Spier, 1996). Alternatively, resident APCs may be transfected directly and the antigen that is expressed by transfected myocytes is an irrelevant side-product. In either case, the antigen-expressing bone marrow-derived APCs then migrate to lymph nodes where they activate the T and B lymphocytes. Transfected myocytes may also serve as plasmid-depots for continued APC-transfection. Because myocytes expressing the antigen are subject to CTL-lysis (Davis H L, 1997), plasmid released from these myocytes may be picked up by monocytes migrating through the muscle (Wolfgang W. Leitner H. Y., 1999).
Both bone marrow-derived APC (BM-APC) and plasmid-carrying macrophages from the injected muscle to lymph nodes has been observed (Wolfgang W. Leitner H. Y., 1999). The crucial role of DC is also supported by the observation that subcutaneous DNA-injection is very inefficient since this tissue lacks Langerhans cells (Condon C, 1998).
In contrast to muscle, skin has important immunological functions as it represents the ‘first line of defence’ of the immune system. Throughout the epidermis, specialized DC form a 3-dimensional network to assure tight immune-surveillance of the skin. Infections agents stimulate DC to pick up antigen and after migrating to the local lymph nodes initiate an immune response (MC, 1997). The main methods of plasmid-DNA delivery to the skin, by needle injection or by gene-gun, differ in several respects. While needle injection requires relatively large amounts of plasmid (similar to the 50-100 μg dose used in intramuscular immunization), the amount of plasmid required for gene-gun immunization has been titrated down to a few nanogram (Degano P, 1998). As in myocyte-transfection after intramuscular immunization, plasmid can be actively taken up by skin cells but only few cells are transfected after intradermal injection (Wolfgang W. Leitner H. Y., 1999).
When delivered by gene-gun, the plasmid solubilizes when the plasmid-coated gold bullet penetrates the cells in the skin. Thus, plasmid is directly deposited into cells transfecting up to 20% of the cells in the target-area (Williams R S, 1991). Tissue stress resulting from the blast may contribute to the activation of DC. Indeed, the total number of DC in the skin-draining lymphoid tissue increases enormously after gene-gun immunization, although the majority of these cells do not carry the plasmid. The small amounts of immunostimulatory DNA delivered with the gene gun may not be sufficient to mediate a Th1-type response allowing Th2-type responses to emerge (Barry M A, 1997). By modifying the immunization-regimen, either IgG1 or mixed IgG1/IgG2-responses can be induced by gene-gun immunization (Leitner W W, 1997). Furthermore, a gene gun-induced Th2-type response can be switched to a Th1-type response by co-delivering the genes for IL-2, -7 or -12 (Prayaga S K, 1997) (see FIG. 3).
Improving the Efficacy of DNA Vaccines
A large number of approaches have been used in an attempt to improve the often poor efficacy of DNA vaccines. Because the efficacy of DNA vaccines in many systems has not been satisfactory, the most simple and unexpectedly effective strategy is increasing the intervals between immunizations and, thereby, the ‘rest-period’ of the immune system. In addition, many elements of the plasmid can be optimized for use of the vector as a DNA vaccine (Wolfgang W. Leitner H. Y., 1999). Based on the idea that more antigen is better, most DNA vaccines use strong viral promoters and are geared towards maximum expression. Other sequences that can be optimized in a plasmid include introns, enhancers and poly-adenylation signals, see, e.g. (Bohm W, 1996), (Danko I, 1997) and (Montgomery D L, 1993). Without a doubt, one of the outstanding features of DNA immunization is the opportunity to co-deliver information together with to the antigen-coding sequence on the plasmid DNA. In recent years several strategies have successfully been used to modulate the immune response after DNA immunization, such as: (i) different modes and sites of gene delivery, (ii) co-delivery of genes or adjuvant molecules with regulatory and/or stimulatory properties and (iii) modification of the vector sequences by inserting or deleting cytosolic or endosomal transport signals.
To improve the immunogenicity of an antigen encoded by a genetic vaccine, functional sequences like the intracellular domain or the trans-membrane sequence can be eliminated (Chen Y, 1998). Antigens can be targeted to the Class-I or Class-II processing pathways with the addition of sequences designed to direct intracellular trafficking. Finally, immunodominant epitopes from antigens can be expressed as minigenes, or they can be buried within unrelated, but highly immunogenic core-sequences (Ciernik I F, 1996). This may be especially useful in cases where ‘full-length’ proteins are not suitable as vaccine candidates, because they are toxic for the host or immunosuppressive. Antigenic proteins can be maximally truncated, leaving only defined epitopes for B or T cells. Antigens consisting of CD8+ T cell-epitopes alone are sufficient to induce CTLs as this approach has also successfully been used for CD4+-T cell epitopes (Casares S, 1997). To overcome MHC-restriction of individual epitopes or to induce a broader range of effector-cells, it is possible to deliver multiple contiguous minimal epitopes in form of a ‘polytope’. To improve MHC-1-loading, endoplasmic reticulum (ER) insertion signal sequences can be attached to minigenes. These sequences can facilitate the targeting of the antigen to the ER, where MHC-I molecules are complexed with antigen. This approach was pioneered in the vaccinia system and also works for peptide-immunization (Minev B R, 1994). An adenovirus leader-sequences has successfully been used to target DNA vaccine encoded CD8+ T cell epitopes to the ER (Ciernik I F, 1996).
Helper epitopes, such as the hepatitis B core-antigen, can activate B cells and elicit strong T cells responses adding significantly to DNA-based vaccines against hepatitis (Kuhrober A, 1997).
Cytokines, such as IL-2, IL-6, IL-7 and especially IL-12 can significantly improve vaccine-induced immune responses, accelerating and augmenting it as well as directing it, for example, towards a Th1- or Th2-type response (Irvine K R, 1996). Other useful cytokine adjuvants include GM-CSF, a cytokine thought to recruit and mature dendritic cells (Mellstedt H, 1999). Besides using exogenous factors, cytokines as well as chemokines encoded on plasmid DNA or as cDNA have been used to study, modulate or enhance a DNA vaccine induced immune response. One study shows the conversion of a non-immunogenic antigen into a DNA vaccine by fusing it to the genes for chemokines (Biragyn A, 1999).
To develop a T cell response, APCs have to deliver two signals to the T cell: one signal is from the MHC/peptide complex to the T cell receptor, the second is from a costimulatory molecule, of which B7 is perhaps the most important and best characterized. In the absence of costimulation, T cells may become anergic preventing self-reactive cells from producing auto-reactivity. B7.1 and B7.2 are expressed on professional APC and on a variety of other tissues after exposure to inflammatory cytokines (Pechhold K, 1997).
Co-delivery of B7.1 with a malaria antigen (Pb-CSP) by gene gun significantly increased the protective effect of a low-expressing plasmid, but not of a high expresser plasmid (Wolfgang W. Leitner H. Y., 1999)
Non-methylated, palindromic DNA-sequences containing CpG-oligodinucleotides (CpG-ODN) can activate an ‘innate’ immune response by activating monocytes, NK cells, dendritic cells and B-cells in an antigen-independent manner (immunostimulatory DNA sequences, ISS) (Wolfgang W. Leitner H. Y., 1999). CpG-ODN have been reported by one group to be as effective an adjuvant as Complete Freund's Adjuvant and to be without significant toxicity (Weiner G J, 1997). In addition, CpG motifs enhance the expression of various co-stimulatory ligands such as CD80, CD40, and ICAM-1 on APCs. In the case of DNA vaccines they can either be co-administered with plasmid-DNA in the form of oligonucleotides or the number of ISS on the plasmid-backbone can be increased.
The delivery of the same antigen multiple times using carriers with little or no immunogenic crossreactivity (heterologous prime-boost-regimen) provides several advantages over the repeated delivery of an antigen with the same carrier (homologous boosting). The repeated use of any given recombinant virus-based vaccine may be impaired by anamnestic responses to the carrier itself (Restifo N, 1999). Including DNA in these regimens may also shift the response towards Th1, even when a Th2-type response was initiated with recombinant protein (Li X, 1998). Heterologous boosting yielded full protection in the P. berghei malaria model when plasmid immunization was followed by administration of recombinant vaccinia virus. Homologous boosting was weak or ineffective in this model (Sedegah M, 1998).
‘Self-replicating’ genetic vaccines are designed to overcome the poor efficacy of some current DNA-based and RNA-based genetic vaccines. The idea and the elements for this new generation of vaccines come from members of the Alphavirus genus, which includes Sindbis virus, Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEE) virus. These RNA viruses contain a single copy of positive-stranded RNA encapsidated by a protein/lipid envelope. The viral RNA encodes its own RNA replicase, an autoproteolytic polyprotein that cleaves itself into four non-structural protein components (nsP1-4) (Berglund P, 1996). Upon infecting a cell, the viral RNA first translates the replicase complex, which in turn drives its own RNA replication. The replicase complex then synthesizes a genomic negative-strand (anti-sense RNA), which is used as a template for the synthesis of the genomic positive-stand RNA as well as a subgenomic RNA encoding the structural viral proteins (see FIG. 4). The genes for structural proteins can be replaced with the gene for the antigen of interest to construct powerful replicase-based vaccines (Caley I J, 1997).
Theoretically up to 200,000 copies of RNA can be produced in a single cell within 4 h and expression of the encoded antigen can be as much as 25% of total cell protein (Rolls M M, 1994). The alphavirus replicase functions in a broad range of host cells (mammalian, avian, reptilian, amphibian and insect cells) (Rolls M M, 1994). Replication takes place in the cytoplasm of the host cell and, therefore, is independent of the host's replication system. All the above features, i.e. high level expression, broad host range and cytoplasmic replication, are useful features in genetic vaccine development.
To facilitate vaccine production, genomic alphavirus RNA alone can be used as a vaccine vehicle. The in vitro transcribed self-replicating RNA contains sequences coding for the SFV replicase and a model antigen. DNA-based vaccines can also be constructed by inserting a strong promoter like the human CMV immediate promoter/enhancer element to initiate the transcription of the full length ‘genomic’ RNA in the nucleus (Tubulekas I, 1997). Replicase-based DNA vaccines may be significantly more immunogenic and efficacious than conventional DNA-plasmid vaccines when low doses of the vaccine are given. Indeed, nanogram amounts of replicase-based vaccine can induce antigen-specific antibody and CD8+ T cell responses (Wolfgang W. Leitner H. Y., 1999).
Furthermore, it has been demonstrated that inclusion of the CMV intron A improved the level of expression of transgenes expressed by the CMV promoter or other promoter/enhancers (Chapman B S, 1991). However, some widely used virally-derived promoters, such as the CMV promoter, may not be suitable for some gene therapy applications since treatment with interferon-γ or tumour necrosis factor-α may inhibit transgene expression from DNA vaccines containing these promoters (Harms J S, 1999). Thus, alternatives to the CMV promoter have been sought. For example, the desmin promoter/enhancer, which controls expression of the muscle-specific cytoskeletal protein desmin, was used effectively to drive expression of the hepatitis B surface antigen priming both humoral and cellular immunity against the antigen. These responses were shown to be of a comparable magnitude to those in mice immunised with comparable DNA vaccines containing the CMV promoter. Other tissue-specific promoters that have been studied include the creatine kinase promoter, also specific to muscle cells and the metallothionein and 1,24-vitaminD(3)(OH)(2) dehydroxylase promoters, both of which are specific to keratinocytes (Itai K, 2001).
Since the rate of transcriptional initiation is generally increased by the use of strong promoter/enhancers, the rate of transcriptional termination may become rate-limiting (NJ, 1989). In addition, the efficiency of primary RNA transcript processing and polyadenylation is known to vary between the polyadenylation sequences of different genes. Thus, the polyadenylation sequence used within a DNA vaccine may also have significant effects on transgene expression. For example, it was demonstrated that the commonly used SV40 polyadenylation sequence was less efficient than the minimal rabbit β-globin and bovine growth hormone polyadenylation sequences in mouse liver, although addition of a second SV40 enhancer downstream of the SV40 polyadenylation signal did increase expression to a level comparable to the other signals (Xu Z-L, 2001). Therefore, it is possible that the strategy of inserting a second SV40 enhancer downstream of a SV40 polyadenylation sequence may be utilised in the construction of more efficient vectors.
Sequences flanking the AUG initiator codon within mRNA influence its recognition by eukaryotic ribosomes. As a result of studying the conditions required for optimal translational efficiency of expressed mammalian genes, the ‘Kozak’ consensus sequence has been shown to be important (M, 1997). It has been proposed that this defined translational initiating sequence (−6 GCCA/GCCAUGG+4) should be included in vertebrate mRNAs located around the initiator codon (M, 1997). It has also been suggested that efficient translation is obtained when the −3 position contains a purine base or, in the absence of a purine base, a guanine is positioned at +4 (M, 1997). Prokaryotic genes and some eukaryotic genes do not possess Kozak sequences. Therefore, the expression level of these genes might be increased by the insertion of a Kozak sequence.
The presence of a TPA signal sequence facilitates the secretion of the antigens from the host cells. Consequently, the enhanced protection seen for these TPA-positive plasmids may result from their capacity to induce higher concentrations of other critical cytokines or chemokines (Flynn, 1995).
Adhesion molecules may also serve as potent candidates for immunomodulation by co-injection of the respective genes together with plasmid DNA (J J, 1999). Intracellular adhesion molecule-1 (ICAM-1), lymphocyte function associated-3 (IA-3), and vascular cell adhesion molecule-1 (VCAM-1) along with DNA immunogens have been used for immunomodulation. Antigen-specific lymphoproliferation and cytotoxic responses were enhanced by co-expression of ICAM-1 and LA-3.
Yet another strategy for deliberately modulating intracellular events was the co-delivery of ubiquitin in the form of a fusion protein. In contrast to the effects induced by the leader sequences, tagging proteins with this or similar sequences accelerates and increases the proteasomal degradation. While this approach will improve the induction of CTL responses it will also drastically decrease the antibody response (Fu T M, 1998).
DNA Vaccination Based on the CSP
DNA vaccination based on the circumsporozoite protein (CSP) gene is sufficient to protect at least 90% of BALB/c (Elke S. Bergmann-Leitner, 2005) and C57BL/6 mice against Plasmodium berghei sporozoite infection. This protection depends on epidermal injection of plasmid DNA with a gene gun (Leitner W W, 1997), immunization intervals that optimize the induction of Th2-type immunity (Weiss, 2000), and removal of the glycosyl-phosphatidylinositol (GPI) signal sequence to allow protein secretion (Scheiblhofer, 2001). By applying these principles, researchers are able to protect at least 90% of vaccinated mice against sporozoite challenge following two or three immunizations with the DNA vaccine CSP(−A). Of greater value would be a regimen that induces protective immunity after a single immunization. For a malaria vaccine to be useful in the field, protection against infection needs to be provided after a minimum number of immunizations, optimally a single delivery.
DNA Vaccination Using C3d
A variety of factors and signals initiate and enhance B cell APC function and thus represent potential adjuvants. Among these is C3d, which is generated from C3 during complement activation, and binds to complement receptor 2 (CR2, CD21), on mature B cells. C3d-mediated B cell activation occurs as a consequence of simultaneous engagement of the antigen-specific surface-bound Ig and CD21 by antigen/C3d complexes (Tsokos, 1990). It has been demonstrated, that as an adjuvant, C3d can also function independently of CD21/CD35 (Haas, 2004).
The cleavage of the third complement protein, C3, that is mediated by the C3 convertases of the classical or alternative pathways of complement activation A is the most critical reaction in the complement system (Weis, 1984). The major cleavage fragment, C3b, covalently attaches to the antigen-antibody complex or bacterium bearing the C3 convertase by a transacylation reaction involving the glutamyl component of an internal thiolester (Weis, 1984). This C3b may then be hydrolyzed by factor I in the presence of cofactors to yield sequentially iC3b and C3d,g, which remain attached to the target via the covalent binding site. C3d,g is susceptible to further proteolysis by noncomplement-derived enzymes such as trypsin and neutrophil elastase to generate the C3d fragment (Lachman, 1982).
These proteolytically generated fragments of C3 mediate the binding of complement-activating complexes to various cell types involved in immune and inflammatory reactions by interacting with three types of cellular receptors. The C3b receptor, also termed CR1, has primary specificity for C3b but may also bind iC3b and C4b, the major cleavage fragment of C4, and is present on erythrocytes, neutrophils, monocytes/macrophages, B cells, some T cells, and glomerular podocytes (Weis, 1984). CR1 has been shown to be a membrane glycoprotein having two allotypic forms of 250,000 Mr and 260,000 Mr. A receptor for iC3b, known as CR3, binds this cleavage fragment of C3 and possibly C3d,g and is expressed by neutrophils, monocytes/macrophages, and large, granular lymphocytes having natural killing and antibody-dependent cell-mediated cytotoxic activity (Carlo, 1979). CR3 has been identified as a glycoprotein having two polypeptide chains of 185,000 Mr and 105,000 Mr, respectively (Wright, 1983). An additional type of receptor, designated the C3d receptor or CR2, binds the C3d region of iC3b and C3d,g and is found only on B lymphocytes and certain B-cell lines, such as the Burkitt lymphoma line Raji (Ross G. R., 1973).
C3d expresses an antigenic determinant, detectable by monoclonal antibody (mAb) 130 which is also expressed by iC3b and C3dg but not by C3 or C3b (Tamerius, 1985). Thus, both the CR2 binding site and the neoantigenic site detected by mAb 130 are located in C3d, which also contains the site for covalent binding of membrane and which remains bound to the target cell or particle after removal of C3c by factor I.
Fusion of two or three tandem copies of C3d to recombinant hen egg lysozyme (HEL) has been shown to enhance dramatically the immune response to HEL (Dempsey, 1996). This approach was subsequently used successfully to enhance the efficacy of DNA vaccines. Indeed, constructs encoding genes for pathogen-derived antigens linked in tandem to sequences encoding for C3d delivered more protective immune response than constructs without the C3d (Elke S. Bergmann-Leitner, 2005).
The potential adjuvant effect of C3d in a DNA vaccine against P. berghei malaria was studied using a previously established CSP-based DNA vaccine known as CSP(−A) (Elke S. Bergmann-Leitner, 2005). Paradoxically, a significant reduction of protection against a challenge with sporozoites when two copies of C3d were attached to the CSP(−A) gene was observed, even after three immunizations. It has been determined that C3d associates with the C-terminus of CSP, shifts the immune response to a Th1-type response, limits the humoral response against C-terminal epitopes and significantly reduces protection against P. berghei infection (Elke S. Bergmann-Leitner, 2005). Therefore, the fusion of antigens to C3d is not a generally applicable adjuvant strategy.
It is known generally that the production of high molecular weight polypeptides containing multiple repeating sequences is difficult because of the tendency of repeated DNA sequences to undergo rearrangement during replication. Some of the limitations on internal repetitiveness in plasmids have been discussed by Gupta (Bio/Technology 1. 602-609, 1983). Ferrari et al (U.S. Pat. No. 5,641,648) have described methods for expression of repetitive sequences using synthetic genes constructed from monomeric units which are concatenated by ligation. DNA sequences encoding the same repeated amino acid sequence but differing in nucleotide sequence either within or between monomers were constructed by exploiting the redundancy of the genetic code. The resulting lack of precise repetitiveness at the nucleotide level reduced homologous recombination to the point where the repeated oligopeptide sequence could be expressed. The work of Ferrari et al was restricted to relatively short repeating units of 4 to 30 codons (amino-acids) repeated a large number of times (typically about 30-fold).
Using p28 of the CR2 Binding Motif of C3d as a Molecular Aduvant
Complement fragment C3d fused to hen egg lysozyme has been shown to generate a robust antibody response (Dempsey, 1996). It was proposed that direct targeting of the fusion protein to the complement receptor 2 (CR2) was responsible for the observed effect. Recent reports have indicated that enhancement of the immune response by C3d containing immunogen constructs is accomplished by additional pathways because C3d can act as adjuvant even in the absence of CD21/CD35 (Haas, 2004). Using C3d as a fusion partner, we had sought to further improve the efficacy of a malaria DNA vaccine encoding the major surface antigen of Plasmodium berghei, circumsporozoite surface antigen (CSP). Unexpectedly, however, immunization with this construct led to loss of protective immunity (Elke S. Bergmann-Leitner, 2005). In addition, immunization with CSP-C3d led to abrogation of the antibody response against the C-terminus of CSP, a shift in the T cell response from Th2 to Th1-type responses and impaired affinity maturation of antibodies specific for CSP. Because we found that C3d binds to the C-terminus of the CSP (Elke S. Bergmann-Leitner, 2005), we considered binding of C3d to CSP to represent an immune escape mechanism whereby sporozoites may evade the production of protective antibody.
In an effort to take advantage of the reported adjuvant potential of C3d, while avoiding a binding of the C3d-adjuvanted protein to immunogenic regions of CSP, we decided to use the CR2 binding motif of C3d, known as p28, as a molecular adjuvant to CSP which, in theory, should provide the immune-enhancing effects of CR2-biding without masking immunogenic regions of CSP. p28 tetramers have been shown before to enhance the anti-Ig-induced B cell response (Tsokos, 1990). We demonstrated that immunization of mice with CSP-3 p28 DNA constructs provides better protection than CSP alone constructs 6 weeks after the 2nd boost and this protection is associated with better IgG1 anti-P. berghei antibody production and absence of IgG2a anti-P. berghei antibody. In contrast, immunization with CSP-3 C3d DNA constructs resulted in limited protection, failure to produce IgG1 antibody and robust production of IgG2a antibody. Therefore, use of p28 rather the full length C3d may serve as a more reliable molecular adjuvant.
Characterization of p28
CR2 is a 140-kDa glycoprotein that specifically binds iC3b, C3dg. and C3d fragments of C3 and the EBV envelope protein gp350/220 (Tsokos, 1990). CR2 is expressed primarily by B cells, although it has been found on other cell types (Tsokos, 1990).
CR2 binds a site on C3 that is composed of residues 1205-1124 of the C3 sequence (JOHN D. LAMBRIS, 1985). Synthetic C3 peptides P14 (residues 1201-1214) and P28 (residues 1187-1247) bind to CR2 (JOHN D. LAMBRIS, 1985) and can be used as ligands in functional assays. A number of murine mAb that bind human CR2, including the murinem Ab HB5, have been produced (Ross G. a., 1985).
CR2 has been considered to play a role in B cell differentiation and proliferation. Polyclonal and monoclonal antibodies to CR2 as well as particle bound C3d have been shown to enhance B cell responses in different systems (Tsokos, 1990). P14 and P28 peptides inhibit the maturation of murine B cell progenitors (Gisler, 1988) while they support the growth of CR2-binding EBV lymphoblastoid B cell lines (Servic, 1989).
Despite all efforts to generate a vaccine that induces an immune response against a malaria antigenic determinant and protects from illnesses caused by the malaria parasite, many vaccines do not fulfill all requirements as described above. Whereas some vaccines fail to give a protective efficiency of over 85% in vaccinated individuals, others perform poorly in areas, such as, production or delivery to the correct cells of the host immune system.
Therefore, there exists a need for a more reliable molecular adjuvant for DNA vaccinations against Malaria.